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Development of recombinant proteins with an aim to increase their therapeutic activity by the regulation of intracellular sorting

开发重组蛋白，旨在通过调节细胞内分选来提高其治疗活性

基本信息

批准号：
10557230
负责人：
SUGIYAMA Yuichi
金额：
$ 8.06万
依托单位：
The University of Tokyo
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
1998
资助国家：
日本
起止时间：
1998 至 1999
项目状态：
已结题

项目摘要

Biologically active proteins usually exhibit the short plasma half-life, repeated and high doses being necessary to obtain their pharmacological activity in vivo. Since their clearance mechanism is the receptor-mediated endocytosis and subsequent lysosomal degradation in target organs, the aim of this study is to regulate their intracellular sorting in order to avoid such degradation pathway. Hepatocyte growth factor (HGF) was fused with endoplasmic reticulum retention signal and nuclear localization signal by gene recombinant techniques. HGF genes obtained in this way were inserted into PUC-SRaipha vector and transfected into COS-7 cells. HGF produced in the medium was dialyzed and purified by heparin-affinity chromatography and reverse phase HPLC. HGF was iodinated by the chloramine-T method, its molecular weight being checked by SDS-PAGE. The biological activity of each HGF derivative was checked by assessing the stimulatory effect on DNA synthesis in primary cultured rat hepatocytes. After incubating with cell lines which highly express HGF receptor, the trichloroacetic acid-soluble radioactivity in the medium gradually increased after a lag-time. However, the increase in TCA-soluble radioactivity during the incubation period with radiolabeled HGF mutant was much lower, indicating that its intracellular degradation is much slower than the wild-type HGF. Thus, the regulation of the intracellular fate of the endocytosed ligand can be, at least partially, regulated by the insertion of a certain type of signal sequences. However, the stability of such a mutant in the medium was not so much increased probably because of the low efficiency in the recycle of the ligand. Therrefore, further improvement has to be considered to change the sorting after avoiding lysosomal degradation. The dissociation kinetics in the endosomal compartment may be the other target to improve the efficiency of ligand recycle.

生物活性蛋白通常具有较短的血浆半衰期，在体内获得药理活性需要重复和高剂量。由于它们的清除机制是受体介导的内吞作用和随后在靶器官中的溶酶体降解，因此本研究的目的是调节它们在细胞内的分选，以避免这种降解途径。利用基因重组技术将肝细胞生长因子(HGF)与内质网滞留信号和核定位信号进行融合。将获得的HGF基因插入到pUC-SRaipha载体中，并将其导入COS-7细胞。用肝素亲和层析和反相高效液相色谱法对培养基产HGF进行透析和纯化。用氯胺-T法对HGF进行碘化，用SDS-PAGE检测其相对分子质量。通过评估HGF衍生物对原代培养大鼠肝细胞DNA合成的刺激作用来检测其生物活性。在与高表达HGF受体的细胞株孵育后，培养液中的三氯乙酸可溶性放射性在一段滞后时间后逐渐增加。然而，放射性标记的HGF突变体在孵育期间TCA可溶性放射性的增加要低得多，这表明它的细胞内降解比野生型HGF慢得多。因此，内吞配体的细胞内命运的调节可以至少部分地通过插入特定类型的信号序列来调节。然而，这种突变体在培养基中的稳定性并没有得到很大的提高，这可能是由于配体回收效率较低所致。因此，在避免溶酶体降解后，必须考虑进一步改进以改变分选方式。内质体室内的解离动力学可能是提高配体循环效率的另一个目标。